Summary During sperm formation, the quantity of many centrosomal proteins declines in a process known as centrosome reduction. However, the role and importance of this reduction in sperm differentiation is poorly understood. In fact, centrosome reduction is often discounted on the assumption that it is inconsequential for male fertility. However, this dogma has not been rigorously tested and our data argues differently. Therefore, the demonstration that centrosome reduction is mediated by specific pathways and is essential for post- fertilization embryo development has the potential to transform the field. Consequently, the long-term goal of the proposed research is to reveal both the biochemical pathways underlying centrosome reduction during sperm maturation and the potential involvement of centrosome reduction in male fertility. The objective of this application is to perform a pilot study in Drosophila melanogaster that will generate tools essential for a detailed follow-up investigation of centrosome reduction. Drosophila is an excellent genetic model to begin these studies because, like in humans and many other animals, specific centrosomal proteins are reduced at distinct steps during sperm differentiation, and these modified sperm centrosomes are critical for zygotic function after fertilization. Indeed, data from the Avidor- Reiss lab indicates that a specific, kinase-dependent pathway is essential for the reduction of one conserved centrosomal protein, but not others, and that blocking this reduction interferes with post- fertilization development. Therefore, our central hypothesis is that decreases in specific centrosomal proteins during centrosome reduction are essential for the distinct role of the zygotic centrosome in post- fertilization embryo development. Our rationale is that the identification of key genes/proteins involved in Drosophila centrosome reduction will allow us to better define the mechanism and function of centrosome reduction in sperm maturation. Our specific aim is to identify genes that regulate the reduction of specific centrosomal proteins during spermiogenesis. To achieve this aim, we will perform a visual RNAi screen targeting kinases and phosphatases that will cause abnormal localization of fluorescently-tagged centrosomal proteins in the spermatozoa. This study is innovative because it is the first to use a genetic approach and genetically-tagged markers to study centrosome reduction. This study is expected to vertically advance the understanding of how and why centrosomal proteins decrease during sperm formation. Ultimately, knowledge gained from this basic research has the potential to inspire research for new causes of male infertility, early stage miscarriages, and developmental diseases.